Abstract

The serotonin transporter (SERT) is an integral membrane protein that exploits preexisting sodium-, chloride-, and potassium ion gradients to catalyze the thermodynamically unfavorable movement of synaptic serotonin into the presynaptic neuron. SERT has garnered significant clinical attention partly because it is the target of multiple psychoactive agents, including the antidepressant paroxetine (Paxil), the most potent selective serotonin reuptake inhibitor known. However, the binding site and orientation of paroxetine in SERT remain controversial. To provide molecular insight, we constructed SERT homology models based on the Drosophila melanogaster dopamine transporter and docked paroxetine to these models. We tested the predicted binding configurations with a combination of radioligand binding and flux assays on wild-type and mutant SERTs. Our data suggest that the orientation of paroxetine, specifically its fluorophenyl ring, in SERT's substrate binding site directly depends on this pocket's charge distribution, and thereby provide an avenue toward understanding and enhancing high-affinity antidepressant activity.

(a,d,e) hsSERT homology model in an outward-open state based on the dmDAT-cocaine complex (PDB ID 4XP4). (a) Side view of the solvent-accessible surface, with the S1 and S2 ligand-binding sites indicated. The protein is portrayed with cartoon helices colored orange (TM3), blue (TM8), magenta (TM10), and gray (all others). TMs 1 and 6 are foremost but not shown for clarity. Approximate membrane boundaries are represented as brown lines. (b) Chemical structure of paroxetine: ((3S,4R)-3-[(2H-1,3-benzodioxol-5-yloxy)methyl]-4-(4-fluorophenyl)piperidine). This SSRI is composed of three functional moieties: piperidine, benzodioxol, and fluorophenyl rings. (c–e) Residues composing the S1 binding site. (c) Schematic of the subsite definitions for both hsSERT and LeuBAT. The equivalent residues in ggSERT are Y135, D138, S376, and S478 (in subsite A); D209, V212, A213, Y216, N217, T479, and G482 (in subsite B); and Y215, F375, F381, E533, T537, and V541 (in subsite C). The corresponding residues in dmSERT are F90, D93, S328, and S429 (in subsite A); D164, M167, G168, Y171, N172, T430, and G433 (in subsite B); and Y170, F327, F333, N484, P488, and I492 (in subsite C). Residues mutated in this study are in bold italics. (d,e) Structural model of the S1 crevice. (d) View from extracellular pathway, as indicated in (a). The three subsites, A, B, and C, are separated by black dashed lines. Subsite C encompasses definitions from both Sorensen et al. and Wang et al., in which residues implicated only in the LeuBAT structure are to the left of the gray dashed line, referred to here as CW. Amino acids mutated in this study are labeled. Cartoon helices are colored as in (a), with TMs 1 and 6 colored red and green, respectively. Key groups are shown as sticks and sodium ions as spheres. (e) Same representation as (d), but viewed from the plane of the membrane. All residues that have been implicated in antagonist recognition within the hsSERT S1 pocket are displayed as sticks and labeled.

For clarity, only TMs 1 (salmon), 3 (orange), 6 (green), 8 (blue), and 10 (magenta) are shown. Amino acids residing in the S1 pocket are illustrated as thin sticks, with their nitrogen and oxygen atoms colored blue and red, respectively, and their carbon atoms colored the same as the helix to which they belong. They are labeled, with the corresponding residues in hsSERT in parentheses. LeuBAT residues whose SERT counterparts were mutated in this study are displayed as thicker sticks with yellow carbon atoms. The carbon atoms of bound and docked paroxetine are cyan, and the fluorine in the fluorophenyl moiety is violet. The best pose of paroxetine docked to LeuBAT is partially transparent. Sodium and chloride ions are depicted as purple and green spheres, respectively. Hydrogen bonds are indicated as black dashed lines.

SERT amino acid sequences aligned with those of the dmDAT-mfc-cocaine complex (PDB ID 4XP4), wild-type dmDAT, and LeuBAT-Δ13 (PDB ID 4MM4). Transmembrane helices are indicated. For clarity, tick marks have been drawn every 10 amino acids. Residues that differ between hsSERT and either dmSERT and/or ggSERT within the S1 site are highlighted in green. The position that exhibits the maximum reciprocal effect on paroxetine potency upon exchange (A169 in hsSERT, D209 in ggSERT, and D164 in dmSERT) is highlighted in yellow with a red asterisk immediately below the position. Residues mutated to Ala in dmDAT to improve thermostability while retaining residual transport activity are highlighted in cyan.

Homology models of SERT homologues and paroxetine (PXT) poses from the largest cluster for (a) hsSERT-WT (14 poses), (b) hsSERT-A169D (9 poses), (c) ggSERT-WT (8 poses), and (d) dmSERT (6 poses). Docked paroxetine is colored as in . Transmembrane helices are illustrated as transparent ribbons and colored as follows: TM1 (salmon), TM3 (light orange), TM6 (pale green), TM8 (blue), and TM10 (magenta). For all complexes, oxygen and nitrogen atoms are red and blue, respectively, while carbon atoms are the same color from the helix to which they belong. Carbon atoms from amino acids targeted for mutagenesis, however, are yellow, and these residues are depicted as thicker sticks. Residues depicted as the thickest sticks , with side chain atoms in partially transparent spheres, are those which, when mutated, had the greatest impact on paroxetine potency (A169D, D209A, and D164A in hsSERT, ggSERT, and dmSERT, respectively). Sodium and chloride ions are shown as purple and green spheres, respectively. Note that S438 and its equivalents in dmSERT (S429) and ggSERT (S478) are behind the docked paroxetine models and thus not visible in all panels. Also note that Y216 (equivalent to Y176 in hsSERT and Y171 in dmSERT) is behind the docked paroxetine poses in the ggSERT image (panel c).